Dihydrophenanthrenes from Juncus effusus as Inhibitors of OAT1 and

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Dihydrophenanthrenes from Juncus ef fusus as Inhibitors of OAT1 and OAT3 Xue Li,† Yilin Qiao,† Xue Wang,† Ruicong Ma,† Tianxiang Li,‡ Youcai Zhang,† and Robert P. Borris*,† †

School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, People’s Republic of China ‡ Tianjin University of Traditional Chinese Medicine, 88 Yuquan Road, Nankai District, Tianjin 300193, People’s Republic of China

J. Nat. Prod. Downloaded from pubs.acs.org by DREXEL UNIV on 03/20/19. For personal use only.

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ABSTRACT: Organic anion transporters 1 (OAT1) and 3 (OAT3) play important roles in the renal elimination of a range of substrate molecules. Little is known about natural products that can modulate OAT1 and OAT3 activities. The medullae of Juncus ef fusus is often used for the treatment of dysuria in traditional Chinese medicine. To study the interactions of phytochemicals in J. ef fusus with human OAT1 and OAT3, a bioactivity guided phytochemical investigation led to seven new phenanthrenoids along with nine known compounds, including eight phenanthrenoids and a benzophenone from the dichloromethane soluble fraction of a methanol extract of the medullae of J. ef f usus. The structures were established by physical data analysis, including highresolution electrospray ionization mass spectrometry and 1D and 2D NMR. The compounds were evaluated for inhibition of OAT1 and OAT3 in vitro. Compounds 10 and 16 were inhibitors for OAT1, and compounds 1−3, 10, and 16 were inhibitors for OAT3 with IC50 values less than 5.0 μM. Dihydrophenanthrene 1 markedly altered the pharmacokinetic parameters of the diuretic drug furosemide, a known substrate of both OAT1 and OAT3, in vivo.

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preliminary studies, the dichloromethane soluble fraction of a methanol extract of J. ef f usus medullae elicited mild inhibition of OAT1 and strong inhibition of OAT3 in vitro and markedly altered the pharmacokinetic parameters of the diuretic furosemide in vivo.4 As part of our continuing efforts to expand the understanding of the inhibition of OATs by phytochemicals in general and TCM in particular, a bioactivity guided fractionation was performed on the extract, followed by structure determination based primarily on liquid chromatography-mass spectrometry (LC-MS) and 1D and 2D NMR, leading to the identification of 16 compounds, including 7 new phenanthrenoids and 9 known compounds. Both in vitro and in vivo bioactivity experiments were performed. This study is the first to evaluate dihydrophenanthrenes as inhibitors of OAT1 and OAT3 and may allow an initial elucidation of the structure−activity relationships within this group. The pharmacokinetics of dihydrophenanthrene 1 on Oats is also described for the first time, which may provide a basis for further drug development.

he organic anion transporters (OATs in humans or Oats in rodents) play key roles in the distribution and excretion of drugs.1 Specifically, organic anion transporter 1 (OAT1) and 3 (OAT3), which are highly expressed in the kidney, play important parts in the renal elimination of a range of substrate molecules.2,3 Both OAT1 and OAT3 are considered to be therapeutic targets for hypertension.4 Oat3 may mediate blood pressure regulation according to data obtained with mice, and an Oat3 inhibitor might be a potential antihypertensive agent.5 Juncus ef f usus L. (“Deng xin cao” in Chinese), belongs to the Juncaceae family and is mainly distributed in wetlands and coastal marshes worldwide. The medullae and the whole herb of J. ef f usus have important applications in traditional Chinese medicine (TCM) for treatment of dysuria, fidgetiness, pharyngitis, aphtha, traumatic bleeding, irritability, and insomnia.6,7 Previous investigations on J. ef f usus resulted in the isolation of phenanthrenoids, flavonoids, coumarins and coumarinic acid esters, terpenoids and terpenoid glycosides, phenolic acids, sterols, and dihydrodibenzoxepin.8,9 Phenanthrenoids appear to be the primary bioactive compounds which have shown cytotoxic,6,7,10 antitumor,11 antialgal,12−14 anxiolytic and sedative,15,16 antimicrobial,17 cellular protective,18 spasmolytic,19 anti-inflammatory,6,20 and phototoxic activities21,22 as well as inhibition of platelet aggregation.23 On the basis of literature reviews, there are no published reports of OAT1 or OAT3 inhibitors in J. ef f usus to date. In our © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The dried medullae of J. ef f usus were extracted with methanol. The methanol-free extract was subjected to standard solvent Received: October 23, 2018

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DOI: 10.1021/acs.jnatprod.8b00888 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Structures of compounds 1−16.

Table 1.

13

C (150 MHz) and 1H (600 MHz) NMR Data of Compounds 1−3 in Methanol-d4 (δ in ppm, J in Hz) 1 δC, type

2 δH

1 2 3 4 5 6 7 8 9 10 11 12 13

122.9, C 157.2, C 114.2, CH 123.9, CH 122.4, CH 139.4, C 136.6, C 130.9, CH 29.4, CH2 26.1, CH2 11.6, CH3 137.8, CH 115.4, CH2

14

171.1, C

119.3, CH2

1a 4a 5a 8a

139.6, 126.4, 127.6, 140.4,

139.2, 127.2, 140.3, 135.6,

6.76, d (8.5) 7.56, d (8.5) 7.84, s

7.74, 2.81, 2.81, 2.19, 7.54, 5.27, 5.70,

s s s s dd (17.5, 10.9) dd (10.9, 1.3) dd (17.5, 1.3)

C C C C

3

δC, type

δH

122.4, C 157.0, C 114.1, CH 124.1, CH 123.0, CH 128.9, CH 130.6, C 139.1, C 27.2, CH2 26.1, CH2 11.5, CH3 172.6, C 136.9, CH

6.74, 7.47, 7.60, 7.67,

d (8.3) dm (8.3) dm (8.1) d (8.1)

2.87, m 2.71, m 2.17, brs 7.12, dd (17.8, 11.4) 5.13, d (17.8) 5.46, d (11.4)

C C C C

δC, type 122.4, C 156.8, C 114.1, CH 124.0, CH 128.2, CH 133.4, Ca 123.0, CH 138.1, C 27.2, CH2 26.2, CH2 11.5, CH3 174.2, Ca 136.9, CH 119.3, CH2 139.0, 127.4, 139.4, 135.4,

δH

6.73, d (8.3) 7.47, d (8.3) 7.59, s 7.59, s 2.88, m 2.72, m 2.18, s 7.11, dd (17.8, 11.3) 5.19, d (17.8) 5.45, d (11.3)

C C C C

a

Assignment was based on HMBC experiment.

H]− m/z 279.1027, 279.1027, and 279.1021, respectively; calcd 279.1021) as well as their 1 H and 13C NMR spectroscopic data, suggesting these three compounds are isomers. The 1H NMR data (Table 1) of compound 1 indicated four aromatic protons, three vinylic protons, two methylenes, and a methyl group. The 13C NMR data (Table 1) consisted of 18 carbon signals, including a carbonyl at δC 171.1, two olefinic carbons at δC 137.8 and 115.4, two methylene carbons at δC

partition as well as a combination of different chromatographic techniques to afford seven new phenanthrenoids, including four dihydrophenanthrenes (1−4) and three phenanthrenes (5−7) together with nine known compounds, including five dihydrophenanthrenes (8−12), two phenanthrenes (13 and 14), one pyrene (15), and one benzophenone (16) (Figure 1). Compounds 1−3 were obtained as yellowish needles with molecular formula C18H16O3 deduced from high-resolution electrospray ionization mass spectrometry (HRESIMS) ([M − B

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Figure 2. Key 1H→13C HMBC (arrow) correlations of 1−7.

Figure 3. Key 1H→1H NOESY (double arrow) of 1−7 and 1H→1H COSY (solid bond) correlations of 1−7.

29.4 and 26.1, and a methyl carbon at δC 11.6. These NMR data confirmed the presence of a 9,10-dihydrophenanthrene skeleton.12 Correlations were observed between δH 6.76 and 7.56 (H-3/H-4) and δH 5.27/5.70 and 7.54 (H-13/H-12) in the COSY spectrum (Figure 3). Key HMBC correlations (Figure 2) from H-11 (δH 2.19) to C-1 (δC 122.9), C-1a (δC 139.6), and C-2 (δC 157.2), from H-3 (δH 6.76) to C-4a (δC 126.4) and C-1 (δC 122.9); and from H-4 (δH 7.56) to C-2 (δC 157.2) and C-1a (δC 139.6), suggested the methyl group was located at C-1, and the downfield chemical shift of C-2 confirmed it as the site of the hydroxy group. The NOESY correlations (Figure 3) between H-11 (δH 2.19) and H-10 (δH 2.81) and between H-8 (δH 7.74) and H-9 (δH 2.81) supported the above distribution, also confirming that the aromatic proton (δH 7.74, s) was attached at C-8. The other aromatic proton (δH 7.84, s) was placed at C-5 in light of HMBC correlations from H-13 (δH 5.27/5.70) to C-6 (δC 139.4), from H-12 (δH 7.54) to C-5 (δC 122.4), from H-5 (δH 7.84) to C-4a (δC 126.4) and C-7 (δC 136.6), and from H-8 (δH 7.74) to C-14 (δC 171.1) and C-9 (δC 29.4), placing the vinylic group at C-6 and the carboxylic group at C-7. From this evidence, the structure of 1 was defined as 7-carboxy-2hydroxy-1-methyl-6-vinyl-9,10-dihydrophenanthrene. The 1H and 13C NMR data (Table 1) of compound 2 indicated that it was also a 9,10-dihydrophenanthrene derivative. The structure of 2 was similar to that of 1 except that the vinylic group was located at C-8 rather than C-6, which was supported by the correlations shown in Figures 2 and 3. In the HMBC spectrum, a terminal olefinic H-14 (δH 5.13) correlated with the signal for C-8 (δC 139.1), and the

proximal olefinic H-13 (δH 7.12) correlated with signals of C-7 (δC 130.6), C-8a (δC 135.6), and C-8 (δC 139.1). The expected COSY correlations between H-3 (δH 6.74) and H-4 (δH 7.47) were observed as well as weak but detectable correlations between each of these protons and H3-11 (δH 2.17) and H2-10 (δH 2.71). Similarly, the expected COSY correlations between H-5 (δH 7.60) and H-6 (δH 7.67) were observed as well as weak but detectable correlations between each of these protons and H2-9 (δH 2.87). Long range coupling correlations like these have not been reported for compounds in this series. H-9 (δH 2.87) also displayed NOESY correlations with the olefinic H-14 (δH 5.13) and H-13 (δH 7.12). Thus, the structure 2 was defined as 7-carboxy-2hydroxy-1-methyl-8-vinyl-9,10-dihydrophenanthrene. The structure of compound 3 was quite similar to that of 2, but the carboxylic acid moiety has shifted from C-7 to C-6. The NOESY data (Figure 3) showed correlations of H-5 (δH 7.59) with H-4 (δH 7.47), H-9 (δH 2.88) with H-13 (δH 7.11), and H-9 (δH 2.88) with H-14 (δH 5.19), while the HMBC data (Figure 2) showed correlations from H-5 (δH 7.59) to C-4a (δC 127.4), C-5a (δC 139.4) and C-12 (174.2), and from H-7 (δH 7.59) to C-6 (δC 133.4) and C-8a (δC 135.4), indicating a carboxylic moiety at C-6 and a vinylic group at C-8. Thus, the structure of 3 was assigned as 6-carboxy-2-hydroxy-1-methyl-8vinyl-9,10-dihydrophenanthrene. Compound 4 was obtained as colorless needles. Its molecular formula, C18H18O2, was determined by HRESIMS ([M − H]− m/z 265.1229, calcd 265.1229), and the presence of 18 carbon signals in the 13C NMR spectrum. The 1H and 13 C NMR data (Table 2) were similar to those of 2 except that C

DOI: 10.1021/acs.jnatprod.8b00888 J. Nat. Prod. XXXX, XXX, XXX−XXX

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13

Article

C and 1H NMR Data of Compounds 4−7 (δ in ppm, J in Hz) 4a

5b

δC, type 1 2 3 4 5 6 7 8 9 10 11 12 13

122.3, C 156.1, C 114.0, CH 123.4, CH 123.3, CH 127.3, CH 137.6, C 137.3, C 27.1, CH2 26.2, CH2 11.5, CH3 63.5, CH2 135.4, CH

14

120.9, CH2

1a 4a 5a 8a 2-OCH3 7-OCH3

138.5, 127.9, 136.3, 134.9,

C C C C

δH

6.71, 7.43, 7.56, 7.32,

d d d d

(8.4) (8.4) (8.0) (8.0)

2.82, 2.70, 2.17, 4.61, 6.87,

m m s s dd (17.9, 11.5)

5.26, dd (17.9, 2.0) 5.59, dd (11.5, 2.0)

6c

δC, type 116.7, C 153.4, C 116.9, CH 121.7, CH 121.2, CH 126.2, CH 136.3, C 133.9, C 123.7, CH 122.6, CH 10.8, CH3 61.1, CH2 133.3, CH 121.7, CH2 131.4, 123.3, 129.6, 127.2,

δH

7d

δC, type

7.26, 8.52, 8.62, 7.75,

d d d d

(8.7) (8.7) (8.5) (8.5)

7.99, 7.89, 2.46, 4.67, 7.17,

d (9.4) d (9.4) s s dd (17.9, 11.5)

120.4, C 154.5, C 110.6, CH 126.7, CH 138.8, C 118.3, CH 156.8, C 108.8, CH 127.3, CH 123.5, CH 11.1, CH3 141.5, CH 114.4, CH2

δH

7.23, de 8.66, d (9.3) overlape 7.19, 7.65, 7.90, 2.59, 7.46, 5.43, 5.77,

d (2.8) d (9.2) d (9.2) s dd (17.2, 10.7) dd (10.7, 1.4) dd (17.2, 1.4)

5.44, d (17.9) 5.78, d (11.5) 132.4, Cf 125.4, C 123.8, C 133.1, C 56.3, CH3 55.4, CH3

C C C C

3.95, s 3.95, s

δC, type 120.2, C 155.0, C 110.7, CH 128.2, CH 138.1, C 125.8, C 154.4, C 111.5, CH 127.8, CH 123.0, CH 11.3, CH3 141.0, CH 119.0, CH2

δH

7.24, d (9.4) 9.01, d (9.4)

14.7, CH3

7.26, 7.57, 7.82, 2.53, 7.35, 5.24, 5.71, 2.41,

s d (9.2) d (9.2) s dd (18.0, 11.3) dd (18.0, 1.7) dd (11.3, 1.7) s

133.3, C 126.5, C 124.2, C 132.0, C 56.4, CH3

3.94, s

a

Recorded at 600 MHz in methanol-d4. bRecorded at 600 MHz in DMSO-d6. cRecorded at 400 MHz in CDCl3. dRecorded at 400 MHz in acetone-d6. eAssignment was based on HSQC experiment. Overlap with solvent peak. fAssignment was based on HMBC experiment.

the carboxylic group of 2 was replaced by a hydroxymethyl moiety in compound 4. The singlet at δH 4.61 in the 1H NMR spectrum was ascribed to H-12 based on NOESY correlations (Figure 3) with H-6 (δH 7.32), H-13 (δH 6.87), and H-14 (δH 5.26) and HMBC correlations (Figure 2) with C-6 (δC 127.3), and C-8 (δC 137.3). These observations supported the assignment of the structure of 4 as 2-hydroxy-7-hydroxymethyl-1-methyl-8-vinyl-9,10-dihydrophenanthrene. Compound 5 was isolated as a yellowish amorphous powder. The HRESIMS data of 5 showed an [M − H]− ion at m/z 263.1072 (calcd 263.1072), suggesting a molecular formula of C18H16O2. The spectroscopic data (Table 2) indicated the presence of a phenanthrene skeleton.14 Comparing the NMR data of 5 with those of 4 indicated that compound 5 was the 9,10-dehydro analogue of 4. This assignment is supported by the correlation between two characteristic ortho-coupled aromatic protons H-9 (δH 7.99) and H-10 (δH 7.89) in the COSY data (Figure 3) and the correlations of H-9 (δH 7.99) with H-13 (δH 7.17), H-9 (δH 7.99) with H-14 (δH 5.44), and H-10 (δH 7.89) with H-11 (δH 2.46) in NOESY data (Figure 3). The structure of compound 5 is defined as 2-hydroxy-7hydroxymethyl-1-methyl-8-vinylphenanthrene. Compound 6 was isolated as a yellowish amorphous powder. Its molecular formula was established as C19H18O2 from the deprotonated molecular ion at m/z 277.1235 [M − H]− in the HRESIMS spectrum (calcd 277.1229). The 1H and 13C NMR spectra suggested that it was a phenanthrene derivative similar to 7-hydroxy-2-methoxy-1-methyl-5-vinylphenanthrene previously identified in J. ef f usus24 but with a methoxy group replacing the hydroxy group. The HMBC correlations (Figure 2) observed from the methoxy signal at δH 3.95 to the downfield-shifted C-7 (δC 156.8) indicated C-7 bears one of the methoxy groups. The NOESY correlations (Figure 3) observed from δH 3.95 to H-8 (δH 7.19) and from H-8 (δH

7.19) to H-9 (δH 7.65) also supported the position of this methoxy group at C-7. Thus, the structure of 6 was defined as 2,7-dimethoxy-1-methyl-5-vinylphenanthrene. Compound 7, a pale yellow amorphous powder, gave a deprotonated molecular ion of 277.1227 [M − H]− (calcd 277.1229) in the HRESIMS, consistent with a molecular formula of C19H18O2, isomeric with compound 6. The structure of compound 7 was similar to the reported 7hydroxy-2-methoxy-1-methyl-5-vinylphenanthrene,24 except that a methyl group in 7 replaced H-6 of the reported structure. The methyl proton signal at δH 2.41 was attributed to the 6-methyl group on the basis of its HMBC correlations (Figure 2) to the C-5 (δC 138.1), C-6 (δC 125.8), and C-7 (δC 154.4) as well as its NOESY correlation (Figure 3) to H-13 (δH 5.24). Thus, the structure of 7 was assigned as 7-hydroxy2-methoxy-1,6-dimethyl-5-vinylphenanthrene. The nine known compounds, including 2,7-dihydroxy-1,6dimethyl-5-vinyl-9,10-dihydrophenanthrene (juncusol) (8),12 2,7-dihydroxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene (effusol) (9),12 7-carboxy-2-hydroxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene (10),11 2,7-dihydroxy-5-(1-methoxyethyl)-1methyl-9,10-dihydrophenanthrene (effususol A)25 (11, isolated as a racemic mixture), 2,7-dihydroxy-5-(1-methoxyethyl)-1,8dimethyl-9,10-dihydrophenanthrene (jinflexin A) (12),17 2,7dihydroxy-1,6-dimethyl-5-vinylphenanthrene (dehydrojuncusol) (13),17 2,7-dihydroxy-1-methyl-5-vinylphenanthrene (dehydroeffusol) (14), 26 2,7-dihydroxy-1,6-dimethylpyrene (15),27 and 4,4′-dihydroxy-3,3′-dimethoxybenzophenone (16),28 were identified by comparison of their NMR data with reported data. Of these, 16 is now reported as a metabolite of the Juncaceae. The isolated compounds were evaluated for their inhibitory activities on OAT1 and OAT3 in cell culture (Figure 4 and Table 3). Results showed that compounds 1, 10, and 16 D

DOI: 10.1021/acs.jnatprod.8b00888 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 4. Dose-dependent inhibition of compounds on OAT1- and OAT3-mediated 6-CF uptake.

Table 3. Inhibitory Effects (IC50, μM) of Compounds on OAT-Mediated Uptake of 6-CF OAT1 OAT3

1

2

3

8

9

10

11

16

8.4 1.3

18.3 1.1

2.8

6.5

23.3 22.7

4.4 1.1

43.1 8.3

3.5 2.3

showed good concentration-dependent inhibition on 6-CF uptake by OAT3 with IC50 < 10 μM. Of these, compounds 10 and 16 exhibited marked inhibitory activities on OAT1 with IC50 value of 4.4 and 3.5 μM, respectively, while compounds 1−3, 10, and 16 displayed strong inhibitory activities on OAT3 with IC50 values of 1.3, 1.1, 2.8, 1.1, and 2.3 μM, respectively. While 16 was observed to be a benzophenone, the other bioactive compounds 1−3 and 10 on OAT1 and/or OAT3 were dihydrophenanthrenes. The corresponding

showed good concentration-dependent inhibition on 6-CF uptake by OAT1 and compounds 1−3, 8, 10, 11, and 16 E

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of OATs, on the one hand, the results of this study provide a rational basis for the therapeutic applications of J. eff usus in TCM or dihydrophenanthrenes in Western medicine. On the other hand, it also showed that taking dihydrophenanthrenes such as 1 or ingesting plants containing these compounds concurrently with drugs that are substrates of the OATs such as furosemide could result in serious interactions.

phenanthrenes were found to have little, if any, activity. It appears likely that the presence of a carbonyl substituent on the dihydrophenanthrene nucleus may contribute greatly to this inhibitory activity on OATs. Based on the above results, dihydrophenanthrenes from J. ef f usus medullae could be used as effective inhibitors or lead compounds for development of inhibitors of OAT1 and/or OAT3. Given the strong inhibitory activity of dihydrophenanthrenes on OATs in vitro and the complex environment in vivo, pharmacokinetic studies of the new dihydrophenanthrene 1 were performed to characterize its interaction with furosemide (FS), a loop diuretic which is a substrate of both OAT1 and OAT3. In our preliminary study, it was found that neither a dose of 20 mg/kg of compound 1 or 10 mg/kg probenecid, the classic inhibitor of OAT1 and OAT3 used here as a positive control, by oral gavage caused any toxic response in rats. The mean plasma concentration−time course curve of FS in rats pretreated with 1, probenecid, and vehicle are shown in Figure 5. Compared with vehicle control, the results indicated that

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on a melting point apparatus (Tianfen Analytical Instrument Factory, Tianjin, China). Optical rotations were measured with an Autopol II automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, United States). All 1D and 2D NMR spectra were acquired using Avance III-400 and 600 MHz spectrometers (BrukerBioSpin, Billerica, MA, United States) operating at ambient temperature. Residual solvent resonances were used as internal standard. Chemical shifts are expressed in δ PPM. UPLC-QQQ-MS data were recorded on an Agilent 1260-6420 series spectrometer (Agilent Technologies, Santa Clara, CA, United States). HRESIMS spectra were performed on a Micro TOF-Q II mass spectrometer (Bruker Daltonics Inc., Billerica, MA, United States) in negative ion mode. Flash chromatography was performed on a Combiflash Rf+ instrument (Teledyne-ISCO, Lincoln, NE, United States) with prepacked silica flash columns (Santai Technologies, Inc., Jiangsu, China) in different sizes. Open column chromatography was carried out using Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden). Analytical HPLC was performed on an Agilent 1260VL quad gradient system (G1311C pump, G1329B autosampler, G1316A thermostated column compartment and G1315D photodiode array detector, Agilent Technologies, Santa Clara CA, United States), using a Waters XSELECT CSH C18 (150 × 2.1 mm, 3.5 μm, Waters Inc., Milford, MA, United States) or an Agilent Poroshell 120 EC-C18 (150 × 2.1 mm, 2.7 μm) chromatographic column. Preparative and semipreparative HPLC separations were performed on an Agilent 1260VL quad gradient system (G1311C pump, and G1315D photodiode array detector) equipped with a Rheodyne 7725i manual injection valve (IDEX Health & Science, Middleboro MA, United States) and Shimadzu CTO-20A column oven (Shimadzu Scientific Instruments, Kyoto, Japan), using a Hypersil Gold C18 chromatography column (250 × 21.2 mm, 5 μm, Thermo Scientific, Waltham, MA, United States and 250 × 10 mm, 5 μm), respectively. All solvents used were HPLC grade (Tianjin Concord Technology Co., Tianjin, China). Anhydrous formic acid and DMSO used were chromatographic grade (Tianjin Guangfu Technology Development Co., Tianjin, China). The determination of fluorescence was performed on a Infinite M200 plate reader (Tecan, Mannedorf, Switzerland) with excitation and emission wavelengths at 485 and 528 nm, respectively. 6-Carboxyfluorescein (6-CF) was obtained from Aladdin (Shanghai, China). Probenecid was purchased from Solarbio (Beijing, China). Poly-D-lysine was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States). Furosemide was purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). Warfarin sodium was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, United States). Bicinchoninic acid (BCA) protein assay kit was purchased from Cwbio (Beijing, China). Hygromycin B was purchased from Solarbio (Beijing, China). Plant Material. The dried medullae of Juncus ef f usus L. (Juncaceae) were purchased from Beijing Tong Ren Tang Health Pharmaceutical Co., Ltd. in Tianjin, China, in October 2016. The plant material was authenticated by Professor Tianxiang Li from Tianjin University of Traditional Chinese Medicine. A voucher specimen (no. 201610) was deposited at the School of Pharmaceutical Science and Technology, Tianjin University. Extraction and Isolation. The dried medullae of J. ef fusus (6 kg) were extracted three times with MeOH (280 L, 1 day each) at room temperature to give the MeOH extract (200 g) on removal of the

Figure 5. Plasma concentration−time course of furosemide (FS) in rats pretreated with compound 1.

pretreatment with probenecid (10 mg/kg) or 1 (20 mg/kg) altered the pharmacokinetics of FS in rats. The AUC0−t (μg/ mL·min) values of FS were increased by 12 and 70% within 10 min, increased by 16 and 62% within 30 min, and increased by 32 and 66% within 90 min after coadministration with oral probenecid and compound 1, respectively. Therefore, concomitant use of 1 with substrates of OAT1 and OAT3 may result in potential clinical interaction. In summary, seven new (1−7) and eight known (8−15) phenanthrenoids and the known benzophenone 16 were isolated from the biologically active dichloromethane-soluble fraction of J. ef f usus medullae via bioactivity guided fractionation. Serial dilution studies established that 9,10dihydrophenanthrenes are the main active constituents of this plant, while the isolated phenanthrenes showed little if any activity. This study also illustrates that medullae of J. ef f usus are a rich source of phenanthrenoids. Evaluation of the inhibitory activity in vitro of these compounds on OAT1 and OAT3 showed that 10 and 16 were strong inhibitors for OAT1, and compounds 1−3, 10, and 16 were strong inhibitors for OAT3. These results represent a first step in exploring the structure−activity relationships within this series of compounds. Additional studies are needed to fully elucidate the SAR of these compounds and explore the structural features responsible for selectivity for OAT1 and/or OAT3. The pharmacokinetic study showed that the oral administration of 1 to rats altered the pharmacokinetics of furosemide in vivo. Given the important physiological and pharmacological roles F

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6-Carboxy-2-hydroxy-1-methyl-8-vinyl-9,10-dihydrophenanthrene (3). Yellowish needles; mp 269−271 °C; [α]17D 0 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 218 (4.14), 288 (3.93) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 279.1021 [M − H]− (calcd for C18H15O3, 279.1021). 2-Hydroxy-7-hydroxymethyl-1-methyl-8-vinyl-9,10-dihydrophenanthrene (4). Colorless needles; mp 226−228 °C; [α]17D −1.45 (c 0.69, MeOH); UV (MeOH) λmax (log ε) 202 (4.53), 219 (4.58), 284 (4.39) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 265.1229 [M − H]− (calcd for C18H17O2, 265.1229). 2-Hydroxy-7-hydroxymethyl-1-methyl-8-vinylphenanthrene (5). Yellowish amorphous powder; UV (MeOH) λmax (log ε) 203 (2.70), 268 (2.73) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 263.1072 [M − H]− (calcd for C18H15O2, 263.1072). 2,7-Dimethoxy-1-methyl-5-vinylphenanthrene (6). Yellowish amorphous powder; UV (MeOH) λmax (log ε) 193 (3.86), 202 (4.09), 218 (4.06), 267 (4.28), 327 (3.34), 344 (3.47) nm; 1H and 13 C NMR, see Table 2; HRESIMS m/z 277.1235 [M − H]− (calcd for C19H17O2, 277.1229). 7-Hydroxy-2-methoxy-1,6-dimethyl-5-vinylphenanthrene (7). Yellowish amorphous powder; UV (MeOH) λmax (log ε) 201 (3.61), 267 (3.62), 333 (2.87), 349 (3.03) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 277.1227 [M − H]− (calcd for C19H17O2, 277.1229). Cell Culture. Human embryonic kidney 293 (HEK293) cell lines stably overexpressing OAT1 and OAT3 were established and identified as previously described.4 Cell lines stably expressing HEK-OAT1 and HEK-OAT3 were obtained by hygromycin B (75 μg/mL) selection and further characterized by both mRNA expression of transporters and their uptake of 6-CF, a fluorescent substrate for both OAT1 and OAT3.29 The cells were cultivated in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 75 μg/mL hygromycin B at 37 °C with 5% CO2. 6-CF Uptake Assay. The 6-CF uptake assay was performed to evaluate inhibitory activity on OAT1 and OAT3 of isolated compounds as previously described.4,30 A density of 5 × 104 cells were seeded per well in 96-well culture plates precoated with poly-Dlysine. Approximately 85% confluency of cells was obtained after growing 24 h. The cells were washed twice and preincubated for 5 min with preheated (37 °C) uptake buffer (135 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 0.8 mM MgSO4, 28 mM glucose, and 13 mM Hepes, pH 7.2) for the following uptake experiments. The uptake buffer containing 4 μM 6-CF in the presence or absence of compounds and probenecid (a classic inhibitor of OAT1 and OAT3, used as a positive control) was incubated for 5 min to conduct uptake. Uptake was terminated by adding 100 μL of icecold uptake buffer, and the cells were quickly washed in each well three times with ice-cold phosphate-buffered saline (PBS). The cells were lysed with 100 μL of 20 mM Tris-HCl containing 0.2% TritonX100. A 50 μL aliquot of lysate was used to quantify fluorescence using a Tecan Infinite M200 plate reader with excitation and emission wavelengths at 485 and 528 nm, respectively. The protein content of the cell lysate was quantified by using a BCA Protein Assay Kit. The intensity of fluorescence was standardized against total protein content, and measured in triplicate. The stock solutions of tested compounds were dissolved in DMSO with a final concentration of 100 mM and dilutions were made using uptake buffer. The compounds which showed more than 50% inhibition for OAT1 or OAT3 at 50 μM were selected for further concentration-dependent inhibition studies. The cells were incubated with uptake buffer containing 6-CF and serial dilution of selected compounds to determine IC50 (50% inhibitory concentration) values. Statistical Analysis. IC50 values shown in Figure 4 and summarized in Table 3 were estimated by nonlinear regression analysis and expressed as mean ± standard error of mean. Statistical analysis was performed using GraphPad Prism version 7.0. For the uptake experiments, data were analyzed with a two-tailed unpaired Student’s t-test. Pharmacokinetic Studies. The experimental protocols and animal handling were performed with approval of the Animal Use

solvent. The MeOH extract was subjected to a standard solvent partition experiment. The sample was dissolved in MeOH and water (500 mL, 9:1 v/v), and extracted with n-hexane (500 mL × 3) to get an n-hexane-soluble fraction. The residual MeOH was removed in vacuo using a rotary evaporator, dispersed in water (500 mL), extracted with CH2Cl2 (500 mL × 3) followed by water-saturated nBuOH (500 mL × 3), to afford a CH2Cl2-soluble fraction, an nBuOH-soluble fraction and an aqueous fraction. After removal of solvent these four fractions were subjected to preliminary evaluation for inhibition of OAT1 and OAT3 in cell culture. The CH2Cl2 fraction elicited mild inhibition of OAT1 and strong inhibition of OAT3 in vitro. The active CH2Cl2 soluble fraction (28.5 g) was fractionated by chromatography on silica gel (1600 g) eluted with a step-gradient of n-hexane, CH2Cl2, and EtOAc (20:8:0, 20:8:1, 20:8:2, 20:8:3, 20:8:8, 0:9:1, 0:7:3, 0:5:5, 0:0:10) to yield 49 fractions which were evaluated for bioactivity against OAT1 and OAT3. Fractions A−H, eluting with n-hexane, CH2Cl2, and EtOAc (0:9:1, 0:7:3, 0:5:5, 0:0:10), were found to have activity in this assay. Fractions A (0.4 g) and B (0.6 g) were combined and chromatographed on a flash silica column (80 g) eluted with a step-gradient of n-hexane, CH2Cl2, and MeOH (20:8:0, 20:8:1, 20:8:2, 20:8:3, 20:8:4) to yield subfractions AB1−5. Subfraction AB2 (0.1 g) was separated on a flash silica column (8 g) eluted by a stepgradient of n-hexane and CH2Cl2 (90:10, 70:30, 60:40, 50:50) to yield 6 (2 mg) and 7 (2 mg). Pooled subfractions AB3−4 (0.8 g) were separated on a Sephadex LH-20 column eluted with (CH2Cl2/ MeOH 1:1) to yield compounds 8 (5 mg), 13 (15 mg), and 15 (8 mg). Fraction C (0.8 g) was chromatographed on a Sephadex LH-20 column (CH2Cl2/MeOH 1:1) to yield 9 (0.5 g). Fractions D (1.2 g), E (1.0 g), and F (1.0 g) were combined and separated on a flash silica column (180 g) eluted by a step-gradient of n-hexane, CH2Cl2, and MeOH (15:8:0, 15:8:1, 15:8:2, 15:8:3) to give a crude compound eluting with a proportion of 15:8:2 on n-hexane, CH2Cl2, and MeOH, and this crude compound was purified further by Sephadex LH-20 (CH2Cl2/MeOH 1:1) to give 14 (2.5 g). Fraction G (1.8 g) was subjected to silica gel flash chromatography (120 g column) eluted with a step-gradient of CH2Cl2 and EtOAc (100:0, 98:2, 96:4, 91:9, 83:17, 50:50, 0:100) to yield subfractions G1−10. Subfractions G3−5 (0.4 g) were pooled and separated by repeated preparative HPLC at 40 °C eluted with 30% aqueous MeCN containing 0.1% formic acid at 8 mL/min and purified by semipreparative HPLC at 30 °C eluted with 28% aqueous MeCN containing 0.1% formic acid at 3 mL/min to afford 1 (75 mg) and 4 (15 mg). Subfraction G6 (0.5 g) was separated by repeated preparative HPLC at 40 °C eluted with 30% aqueous MeCN containing 0.1% formic acid at 8 mL/min to obtain 10 (69 mg) and 2 (10 mg). Fraction H (1.2 g) was subjected to silica gel flash chromatography (80 g column) eluted with a step-gradient of CH2Cl2 and EtOAc (98:2, 96:4, 90:10, 85:15, 50:50, 0:100) to yield subfractions H1−8. Subfraction H3 (0.05 g) was subjected to preparative HPLC at 40 °C, eluted with 20% aqueous MeCN containing 0.1% formic acid at 10 mL/min to give 16 (3 mg). Subfractions H4−5 (0.4 g) were pooled and subjected to preparative HPLC eluting with 28% aqueous MeCN containing 0.1% formic acid at 8 mL/min at 40 °C to give 1 (30 mg), 5 (5 mg), and 10 (40 mg). Subfraction H6 (0.1 g) was subjected to preparative HPLC under the same conditions to give 11(1 mg), 2 (5 mg), 12 (1 mg), and 3 (2 mg). 7-Carboxy-2-hydroxy-1-methyl-6-vinyl-9,10-dihydrophenanthrene (1). Yellowish needles; mp 295−297 °C; [α]16D −2.08 (c 0.48, MeOH); UV (MeOH) λmax (log ε) 195 (3.92), 202 (4.26), 224 (4.30), 260 (4.19), 297 (4.17) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 279.1027 [M − H]− (calcd for C18H15O3, 279.1021). 7-Carboxy-2-hydroxy-1-methyl-8-vinyl-9,10-dihydrophenanthrene (2). Yellowish needles; mp 223−225 °C; [α]16D −2.22 (c 0.45, MeOH); UV (MeOH) λmax (log ε) 202 (4.38), 219 (4.45), 288 (4.26) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 279.1027 [M − H]− (calcd for C18H15O3, 279.1021). G

DOI: 10.1021/acs.jnatprod.8b00888 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Committee (no. 1202) at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences (CAMS, Tianjin, China). The pharmacokinetic study of furosemide (FS) in rats was performed as previous reported with some modification.4,31 Male Wistar rats (200 ± 20 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were housed in the animal facility at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences (CAMS, Tianjin, China) in a temperature controlled room (23 ± 2 °C) with a relative humidity of 55 ± 10%. All rats were acclimated for 1 week, supplied with food and water ad libitum, and then fasted for 12 h prior to the experimental procedure. No toxic response was observed when the rats were administered a dose of 20 mg/kg of 1 or 10 mg/kg probenecid (dissolved in 40% PEG-400) by oral gavage in a preliminary experiment. Three groups of rats with five rats per group were used for the pharmacokinetic study. The rats in experimental group, positive, and negative control groups were pretreated with compound 1 (20 mg/kg), probenecid (10 mg/kg), and vehicle, respectively, by oral gavage. After 5 min, 10 mg/kg FS (dissolved in saline) was administrated by tail vein injection to each group of rats. One milliliter of blood was collected into heparinized tubes through the orbital sinus vein at 2, 5, 10, 15, 30, 60, 90, and 120 min. A 400 μL plasma sample and 1 mL of MeCN (containing 40 μg/mL warfarin sodium as internal standard) were pipetted into a microcentrifuge tube; the mixture was vortexed for 30 min and then centrifuged at 14 000 rpm for 15 min. The supernatant was dried in vacuo and redissolved in 200 μL of MeCN. This solution was centrifuged again, and the supernatant was used for HPLC analysis. Quantification of Furosemide by HPLC. The quantification of furosemide was performed using a modified HPLC method with UV detection.4,32 Chromatographic analysis was performed on an Agilent Poroshell 120 EC-C18 column (150 × 2.1 mm, 2.7 μm) eluted with a mixture of MeCN and ultrapure water (38:62, v/v) containing 0.1% formic acid at a flow rate of 0.2 mL/min and with the UV detection wavelength at 280 nm. The injection volume was 15 μL, and the column temperature was 30 °C. Under these conditions, the retention time of furosemide was 3.85 min, and the retention time of warfarin sodium was 14.52 min.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00888.

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HRESIMS and 1D and 2D NMR spectroscopic data of new compounds 1−7 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel.: +86 183 0224 2039. ORCID

Robert P. Borris: 0000-0002-5317-7382 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Basic Research Program of China (2015CB856500). REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00888 J. Nat. Prod. XXXX, XXX, XXX−XXX